1. Field of the Invention
The present invention is generally related to instrumentation and techniques used for continuously measuring isometric force development during blood clot retraction.
2. Description of the Prior Art
In vivo clotting is known to involve both platelet-mediated, primary, and plasma protein (fibrin)-mediated, secondary, hemostasis. While this artificial separation has allowed simplification of various experimental systems, it is well recognized that neither process occurs as an isolated event. When vascular injury occurs, basement membrane is exposed and platelets adhere thereto. Platelet attachment is followed by attachment of fibrinogen to platelet membrane receptors. When attached to the platelet membrane receptors, fibrinogen serves as a molecular bridge between platelets which promotes further aggregation of platelets at the point of injury. As aggregation proceeds, clot promoting substances are released from the platelet mass which promote formation of fibrin strands. Fibrin reinforces the platelet plug to halt blood loss.
Once the fibrin network is in place, the platelets begin to actively pull on the network strands. The pulling force is directly related to the contraction of microfilaments in the platelet cells. Contraction of the microfilaments is dependent on the intracellular cyclic adenosine monophosphate (cAMP) concentration. The platelets are connected to the fibrin strands by psuedopods; therefore, as the platelets constrict, the fibrin strands are pulled inward. When viewed in vitro, this process, called clot retraction, leads to a dramatic reduction in clot volume. Clot retraction is an irreversible process. It is assumed that clot retraction plays a role in approximating the edges of a tissue defect and in concentrating the clot precisely in the injured area. The platelet-fibrin network subsequently serves as the scaffolding for tissue repair.
Material released from platelets is known to affect fibrin assembly and structure. Platelet Factor 4 (PF4), a protein released from platelet alpha-granules, speeds fibrin clotting and results in thick fiber formation. Thrombospondin, a protein also from the alpha-granules, speeds fibrin formation but causes thinner fiber formation. The addition of platelet extract, prepared by sonicating platelets, to clotting fibrin results in thinner fibers which are more resistant to fibrinolysis. When intact platelets are added to clotting fibrin, thicker fibers are produced and clot dissolution by plasmin is enhanced. The presence of intact platelets also leads to clot retraction and an increased storage elastic modulus.
It is known that intact glycoprotein IIb/IIIa (GPIIb/IIIa) complexes must be present on the platelet surfaces for clot retraction to proceed. Congenital absence of GPIIb/IIIa or blockade of GPIIb/IIIa by synthetic peptide analogues of fibrinogen or by monoclonal antibodies is known to result in absent or reduced clot retraction. In addition, an intact A chain of fibrinogen and adequate calcium are required for clot retraction.
Clot retraction may be inhibited by several mechanisms. High thrombin and/or fibrin concentrations both reduce the extent of clot retraction. Manganesium chloride (MgCl.sub.2) inhibits clot retraction and may operate by competing with calcium. Substances which increase intracellular cAMP such as dibutyryl cAMP (DBcAMP) and prostaglandin E.sub.1 inhibit clot retraction. As noted above, constriction of the microfilaments is dependent on the cAMP concentration. Cytochalsin E and B, which alter filamentous actin, inhibit clot retraction, while colchicine and vinblastine, which disrupt microtubules, have only a minimal effect. Aspirin and indomethacin, which are commonly used antiplatelet agents, do not inhibit clot retraction.
Several techniques have been used to measure clot retraction. The first and most common method is to measure the volume of excluded material and then calculate the percentage of the residual clot volume. This technique, while simple, does not allow measurement of the forces involved in clot retraction nor does it allow detection of the onset of force generation. Other researchers have recorded force measurements by forming cylinders or strips of clot which are subsequently anchored at one end and attached at the other end to a transducer or a mechanical recording device. This technique is reminiscent of studies on smooth muscle contraction and allows suspension of the clot in a bathing solution. Changing the solution provides a way of exposing the clot to a variety of retraction altering agents. Unfortunately, the two end-anchored clot technique does not allow detection of the onset of force development and suffers from the need to manipulate the clot. Clot manipulation can result in "passive" fibrin retraction which is independent of platelet activity. A third technique utilizes a fluids rheometer to measure normal force development during plasma gel clotting and retraction. While the rheological method does allow measurement of both the onset and the kinetics of force development, rheometers are extremely expensive devices which require quite a bit of training to use properly.
Atherosclerosis and malignancy are primary causes of morbidity and mortality in the aging American population. Many complications, perhaps even the pathogenesis of atherosclerosis, are intimately related to clotting. Cerebral vascular accidents and myocardial infarctions are direct results of inappropriate clot deposition. Malignancy is known to be a "hypercoagulable" state associated with increased frequency of deep venous thrombosis and pulmonary embolus. Primary modes of therapy in patients suffering the above conditions involve the prophylactic use of antiplatelet agents or anti-coagulants, or the emergent use of thrombolytics. A need exists for a simple, low cost instrument which can help define the interactions between platelets and fibrin and to assess how these interactions impact on the eventual clot removal. This information is necessary for rational use of available therapy and is essential to the development of newer modes of treatment.